Anti-inflammatory Effects Of Recombinant Interleukin-25 On Acute Ulcerative Colitis

ObjectiveInflammatory Bowel Diseases(IBD)is a collective term for the unknown etiology gastrointestinal disorders such as Crohn's disease(CD)and Ulcerative Colitis(UC) associated with chronic relapsing inflammation.Over recent decades the incidence of IBD has been rising in the world,and IBD will become increasingly common in Asia, as it has been in the last fifty years in Europe and North America.The most recent review of Chinese literature reveals that 143 511 cases of IBD(140 120 of UC and 3391 of CD)were described during the last 15 years,with an 8.5-fold increase during the last 5 years compared with the first 5 years.Although the precise etiology of IBD has not been characterized,the most recent hypothesis concerning the underlying disease mechanism states that individuals who have a genetic predisposition,when confronted with unidentified aggressors from their natural environment,develop a loss of tolerance to luminal bacterial antigens and initiate an uncontrolled inflammatory reaction targeted at the bowel wall and at distant organ systems such as the joints,skin, or biliary tract.Crohn's disease manifests itself as a chronic granulomatous inflammation of the gastrointestinal tract capable of affecting its entire length with the presence of"skip"lesions.Ulcerative colitis,on the contrary,presents as a continuous inflammatory lesion affecting the rectum and colon,lacking granulomatous characteristics.These two forms of IBD appear to show a distinct profiles of T cell mediated immunity.In patients with Crohn's disease,the patterns of cytokines expressed by mucosal lymphocytes generally tend to be consistent with a T-helper-1(Th1)response,including an early increase in the expression of interferon (IFN),IL-2,and IL-12,followed by a subsequent increase in tumor necrosis factor-(?) (TNF-(?))and IL-18.Furthermore,there also appears to be a compensatory increase in IL-10 and transforming growth factor-β1(TGF-β1)levels.In patients with ulcerative colitis,the pattern of cytokine expression differs from that seen in Crohn's disease,with an increased expression of IL-5,IL-6,IL-10,and IL-13.This Th2 response is up-regulated in the colon of UC,although the relevance of elevated Th2 cytokines to the colonic inflammation has not yet been clarified,an increased pro-inflammatory cytokine production observed seems to be related to the inflammation.IL-25/IL-17E is a fifth member of the structurally related IL-17 cytokine family. IL-25 was first discovered in human genomic sequence following analysis of human genomic DNA sequence information available in GenBankTM(accession number CNS01DTR).The human IL-25 gene has been mapped to the region 14q11.2 and spans a region of about 3595bp(according to GenBank accession number NT-026437.Two alternatively spliced mRNA transcripts of the gene encoding two distinct isoforms have been described,the IL-25 isoform 1 precursor(GenBank accession number NM-022789)and the IL-25 isoform 2(GenBank accession number NM-172314).Both mRNAs contain two exons,and isoform 2 lack in internal segment compared to isoform1,leading to a shorter N-terminus.The mRNA isoform 1 is predicted to result into a 177-amino acid protein,while the mRNA for isoform 2 is predicted to encode 161-amino acid protein.The two isoforms are identical in the last 159 carboxy terminal amino acids.A different in the physiological effect of the two isoforms has not yet been detected.IL-25 is secreted as a disulfide linked homodimer of glycoprotein incorporating cysteine residues,with a molecular weight of 34kDa.Structurally,all six family members(IL-17A to IL-17F)conserved cysteine residues that accounting for a characteristic of forming a cysteine-knots and in this respect is related to other better known cytokines such as TGF-βand platelet-derived growth factor.The receptor for IL-25 was first identified as an IL-17B receptor(IL-17BR)also called IL-17 receptor homolog 1(IL-17Rh1)and EVI27.Murine EVI27 was identified through its location at a common site of retroviral integration in BXH2 murine myeloid leukemias.Co-immunoprecipitation experiments have shown that IL-25 is the main ligand for 7BR,while IL-17B demonstrated to interact with 17BR with low affinity.Sequence comparison of the receptors for IL-17A and IL-25 reveals similar overall structure with conservation of several cysteines within the extracellular domains,and also with conserved elements within the intracellular domain,suggesting that these receptors likely engage similar signaling pathways.Indeed,IL-25 was shown to activate NF-κB and to induce the production of chemokine IL-8 in the human renal carcinoma cell line TK-10.IL-17Rh1 shows highest level expression in kidney with moderate expression in multiple other organs,whereas IL-25 mRNA was detected at very low levels in several peripheral tissues,including colon,uterus,stomach,small intestine,kidney,and lung.Native IL-25 expression has been detected from in vitro-differentiated Th2 cells and in vitro-cultured mast cells.However,when the induction of Th2 cytokines was analyzed in vitro,the major cell type in response to IL-25 appears to be a non-T/non-B cell population expressing classâ…¡MHC and CD 11 c molecules,a typical accessory cell phenotype.Treatment of mice with purified IL-25 resulted in the production of cytokines IL-4,IL-5,and IL-13,eosinophilia,increased serum Ig,and the development of striking histological changes in the lungs and the gastrointestinal(GI)tract.Similarly, administration of recombinant IL-25(rIL-25)to mice has been shown to evoke an inflammatory response characterized by the overproduction of Th2 cytokines,hyper production of immunoglobulin IgA and IgE,overproduction of mucus,epithelial cell hyperplasia,and eosinophilia.DSS-induced experimental colitis is widely regarded as a useful murine model of IBD,that reliable for the IBD cellular and molecular studies.The DSS model is easily induced,reproducible,and Th1 cytokine response in acute model reported to resemble that observed in human inflammatory conditions such as IBD.Several animal models for intestinal inflammation have been developed.Oral administration of DSS in drinking water of in several mammals such as mice,rats,guinea pigs and hamsters are able to induce not only an acute,but also a chronic colitis after multiple cycles of DSS. Acute colitis in mice is manifested by diarrhea,weight loss,shortening of the colon, mucosal ulceration,and inflammatory cell infiltration into the left colon.A chronic colitis can be induced in several mammals by feeding them with DSS in their drinking water in cycles of 7 days of DSS followed by 7 days of water.Chronic lesions are marked by increased plasma cells,lymphocytes,macrophages,lymphoid aggregates in the lamina propria as well as the serosa and a patchy distribution of inflammation together with fissuring ulcers.The histopathology of this model resembles to UC and would enable research into the pathogenesis of this disease.Thus,the main objective of this study is to evaluate the effect of rIL-25 on the development of inflammatory Bowel diseases. Methods1.Establishment of UC mouse modelFemale,C57BL/6 mice aged 6-8 weeks,weighed between 18-22g were used in this experiments,they were randomly divided into DSS+rIL-25 treated group,DSS treated group and control group.Acute experimental colitis was induced by oral administration of DSS in drinking water ad libitum for five consecutive days.Mice in the DSS+rIL-25 treated groups and DSS treated group were administered with 2.5% (wt/vol)DSS in drinking water ad libitum while inspected for the development of clinical symptoms,macroscopic changes at autopsy and histological damage.Mice in control group were given free access to normal drinking water.2.Treatment of mice with rIL-25At the start of DSS exposure,mice in the DSS+rIL-25 treated groups were injected i.p.with different concentration of rIL-25 starting from 0.2,0.4 to 0.8μg in PBS respectively.Mice in control group were only injected with PBS.Mice in the DSS treated group were only injected i.p.with 0.1ml of PBS,from day 0 to day 5.Mice in the control group were not given any treatment.Mice were sacrificed on time point basis at day 3 and day 6.3.Assessment of colitisDevelopment and progress of acute colitis was assessed clinically, macroscopically and histologically.Assessment Clinical assessment of DSS-induced colitis were performed in all animal on a daily basis,this including,assessment of mice body weight,evaluation of stool consistency and,visible blood stool.Disease activity index were determined by combining sum of total scores divided by three.Macroscopic assessment of inflammation was performed at autopsy by measuring colon length,and assessing changes in colon gross appearance and presence of colonic blood. Histological assessment was performed on the colon tissue sections and all histological evaluation will be performed in a blinded fashion according morphological criteria grading method.4.Survival studyFor survival study,10 mice from each DSS+rIL-25 treated group and the control group were continuously subjected to DSS until day 7 when they were returned to normal drinking water while assessed for their survival.5.Cytokine assaysELISA technique will be used for measurement of transforming growth factor (TGF)-β1,IL-23,IL-25,IL-17,IL-10,IL-4,and IFN-γ.Spleen supernatants and colonic tissue homogenates samples from each mice group will be analyzed according with provided kit manufactures protocols.The spleens from each treatment group were aseptically removed,placed in 100 mm tissue culture dishes,flushed with PBS supplemented with 2%fetal calf serum(2%FCS PBS)using a 5 ml syringe,and then cells were centrifuged to a pellet at 1,500 rpm.Red blood cells were lysed from spleen samples by incubating cell suspensions for 5 min at 4℃in erythrocyte lysis buffer(170 mM Tris,160 mM NH4Cl,pH 7.4),followed by two washes in 2%FCS PBS at 1,500 rpm.The supernatants were discarded and the cells were resuspended in RPMI 1640 supplemented with 10%FCS.The cells were counted with a hemocytometer,adjusted at 2×10⁶ and then cultured in RPMI 1640 at 37℃in a humidified incubator with 5% CO₂ for 48 hours.Cells were harvested centrifuged and supematants were collected for cytokines analysis.Colon tissue homogenates were prepared for cytokines analysis.the distal colons from each group were collected and homogenized with PBS homogenizing buffer containing 1%Triton X-100 supplemented with cocktail of protease inhibitors.The homogenized solutions were centrifuged at 12,000 rpm for 10 min,and the supematants were separated into aliquots and stored at -70℃.6.RNA extraction and Reverse Transcription-Polymerase Chain Reaction(RT-PCR).Total cellular RNA was isolated from frozen distal colonic tissue by homogenization of the tissue in TRIzol and incubation at room temperature for 5 min. RNA was extracted with chloroform 0.2ml/ml of TRIzol reagent,the samples were shake vigorously,incubated at room temperature for 3 min and then centrifuged for 15 min at 10,000 rpm at 4℃.The aqueous phase was transferred to fresh tubes then precipitated with an equal volume of isopropanol and subsequently centrifuged for 15 min at 10,000 rpm at 4℃.The pellet was washed with 70%ethanol and resuspended in 50μl of DPEC water.Total RNA was determined by spectrometric analysis.RT-PCR was performed in single reaction tubes using the TaKaRa RNA PCR kit.The following primer pairs were used for the PCR:mouse IL-25,5'-GAGGCTTCTGGACTGCGGTGGTCC-3' and 5'-TTGACAG GCGAGGGTCACAGCTCC-3.The RNA reverse transcription was performed in a final volume of 10μl containing 2μl MgCl2(25 mM),1μl 10×RT buffer,3.75μl RNase free water,1μl dNTP mixture(10 mM),0.25μl RNase inhibitor (40/μl),0.5μl Avian Myeloblastosis Virus(AMV)reverse transcriptase(50U/μl),0.5μl random mers(50 pmol/μl),and 1μl positive control RNA.Reactions were incubated at 42℃for 50 min,and then heat-inactivated at 70℃for 15 min.After reverse transcription,the cDNA was aliquoted into tubes that contained specific primer pairs for the mouse IL-25.Briefly,PCR amplifications were performed by addition of 80μl of a master mix containing 10μl 5×RT buffer,28.75μl double distilled water,0.25μl TaKaRa Ex Tag HS 5U/μl,0.5μl Primer 1,0.5μl Primer 2 and 40μl of sample.RNA positive control mixture contained 10μl 5x RT buffer,28.75μl double distilled water, 0.25μl TaKaRa Ex Tag HS 5U/μl,0.5μl control F-1 Primer(20 pmol/μl),and 0.5μl control R-1 Primer(20 pmol/μl).PCR was performed in a thermocycler with an initial denaturation step of 94℃for 5 min,followed by 28 cycles of 1 min at 94℃,1 min at 55℃,and 1 min at 72℃with a final extension at 72℃for 10 min.The amplified PCR products were electrophoresed on a 1%agarose gel together with the 100-bp DNA ladder marker.7.Western blot analysisFrozen distal colonic tissues 100 mg from each group were collected and homogenized with RIPA Lysis Buffer(100mg/ml)supplemented with aprotinin cocktail of inhibitors(0.06μg/ml)and then were incubated at 4℃for 10 min The homogenized solutions were centrifuged at 12,000 rpm at 4℃for 10 min,and the supernatants were separated and the protein concentration in the lysates was quantified with a BCA protein assay kit using bovine serum albumin as a standard.Total protein lysates 20μg per lane were loaded and electrophoresed on a 10%SDS-PAGE before were electro-transferred to Hybond polyvinylidene difluoride membranes at 70 V for 2 h. The membrane was blocked with 5%nonfat milk in Tris-buffered saline supplemented with 0.05%Tween 20(pH 7.6)for 2 h,and incubated with the goat anti-mouse IL-25 polyclonal antibody at a dilution of 1:1000 at 4℃overnight.After washing,membrane was incubated with horseradish peroxidase-conjugated anti-mouse secondary antibody at a dilution of 1:5,000 at room temperature for 2 h.After extensive washing,the blot was developed by using ECL chemiluminescent detection kit according to the manufacturer's instructions and the target protein images were obtained by Bio-Imaging System.8.Immunohistofuorescent stainingSections(5μm)were pretreated by boiling in citrate buffer(pH 6.1)in a microwave. After cooling,non-specific binding was blocked with 5%BSA blocking reagent followed by incubation with primary antibody goat polyclonal anti-mouse IL-25 at a dilution of 1:100 in phosphate buffered saline(PBS)overnight at 4℃in a humidified chamber.As a negative control,sections were incubated with normal goat IgG.After incubation with the primary antibody,sections were washed with three changes of PBS and treated with fluorescein isothiocyanate(FITC)conjugated donkey anti-mouse secondary antibody diluted at 1:50 in PBS for 30 minutes at room temperature in a dark chamber.Thereafter the sections were extensively washed,mounted with aqueous UltraCruz Mounting Medium and then were examined with a fluorescence microscope Olympus BX 61 and images were taken by digital DP 71;Olympus CCD Image system attached to the fluorescence microscope.9.Intracellular flow cytometryIntracellular cytokine staining was performed to assess the production of IL-17 by splenic lymphocytes.Cells were previously stimulated with 50 ng/ml PMA and 750 ng/ml Ionomycin in the presence of Brefeldin A 10μg/ml,for 4.5 h at 37℃in a humidified incubator with 5%CO2.To block Fc receptors,cells were preincubated with 1μg BD Fc Block reagent,for 15 min at 4℃.Cells were harvested and directly stained with surface markers anti-CD4-FITC(RM4-5)and anti-CD8-PE-Cy5 for 30 min at 4℃, followed by two washes in Staining Buffer.Cells were pellet by centrifugation at 2,000 rpm and then resuspended.To proceed with intracellular staining,cells were firstly fixed and permiabilized with BD Cytofix/Cytoperm solution for 20 min at 4℃. Thereafter,cells were washed twice with BD Perm/Wash buffer and then were stained intracellularly with anti-IL-17-PE(TC11-18H10.1)for 30 min at 4℃.Cells were pellet, resuspended in Staining Buffer and analyzed by flow cytometry.Results1.rIL-25 treated mice delayed developed of clinical symptoms and reduced severity of colitis In clinical assessment,DAI was found to be lower in the DSS+rIL-25 treated group than in the DSS treated group(p＜0.05).At autopsy,in macroscopic inspection, the gross colons appearance in the DSS treated mice were found to exhibit typical signs of severe acute inflammation against a mild inflammation exhibited in the DSS+ rIL-25 treated group.Length of colons in the DSS treated group was significantly shortened by 32%while that of the DSS+rIL-25 treated group was shortened by 17% (p＜0.05).In Histological analysis the histological scores in the DSS+rIL-25 treated group was significantly lower compared to that of the DSS treated group(p＜0.05). The DSS treated group revealed grade 4 lesions in contrast to the DSS+rIL-25 treated group revealed grade 2 lesions.2.rIL-25 reduces mice mortality during the development of acute DSS-induced colitisAcute colitis was successively induced all mice by administration of drinking water supplemented with 2.5%DSS for seven days.There was a markedly variation in survival of mice between all groups.Mice treated with 0.4μg rIL-25 shows a significant improvement.The survival of mice treated with 0.2μg rIL-25 was shortened. Our survival study indicates that different dose reproduce distinct effect.3.Expression of IL-25 decreased in DSS-induced colitisWestern-blot and immuno-fluoroscent analysis reveal the expression of IL-25 in the colonic tissues at protein level.IL-25 expression was shown to decrease with increase in severity of colitis.Colon surface epithelium cells were found to be cellular source for IL-25 production.Using RT-PCR technique,we detected the expression of IL-25 at mRNA level.The expression of IL-25 was detected in tissue homogenates by ELISA but not at significant level.4.IL-25 show anti-inflammatory activities on acute DSS-induced colitisThere level of colonic IFN-γ(p＜0.01)was significantly lower in all mice treated with rIL-25 in comparison to the DSS treated mice.Contrarily,colonic elevation of IL-10(p＜0.01),TGF-β1(p＜0.01),and IL-23(p＜0.01)was markedly in the DSS +rIL-25 treated group,but there was no significant difference found in the production of IL-17 between DSS+rIL-25 treated group and DSS treated group.The level of IL-4 was detectable in all experimental groups but at very lower level.Thus,IL-25 reduced acute colitis clinical symptoms and severity by inhibiting or suppressing expression of IFN-γwith subsequent induction of IL-10,TGF-β1 and IL-23 respectively.5.Lower production of IL-17 by CD4+/CD8+T cells in the development of acute DSS-induced colitisWe further performed intracellular cytokine staining to confirm the production of IL-17 by CD4+/CD8+T cells during development of acute colitis.As judged through cytometric fluorescent intensity of intracellular staining,we found lower expression of IL-17 by CD4+/CD8+T cells in both DSS+rIL-25 treated mice and DSS treated mice.DiscussionThe aim of this study was to evaluate the effect of IL-25 on inflammatory bowel diseases.Crohn's disease and ulcerative colitis are the major forms of inflammatory bowel diseases that despite extensive studies etiology behind these diseases remain uncertain.In the present study,firstly we investigated the expression of IL-25 in serum, spleen supernatant,and colonic tissue homogenates as well in the colonic tissue culture supernatant.Our findings reveal expression of IL-25 but at low level.We also performed survival study important to prove and confirm the effect of IL-25 on the development and progress of colon inflammation.Inhibitory effect of IL-25 was proven by low mortality rate observed in the mice treated with IL-25 with improved prolonged survival.That is to demonstrate an improved resistance in DSS toxicity in the colonic mucosal.Interestingly,treatment of IL-25 for three day instead of five was enough to prolong survival time and improve mice health recovery.These findings prove the effective role of IL-25 in the prevention of development of inflammation in colon.The detection of the presence of IL-25 contents on the colon reveals decrease in IL-25 expression in severe colitis.This suggestion was supported with findings in western blot analysis where protein level expression of IL-25 was found high intensity of protein bands on colonic tissue from normal mice but was shown to decrease in severe colitis.Thus this findings show dectrase in IL-25 expression in severe colitis as colonic epithelium cell assumed to be among the colonic cellular source for IL-25In order to evaluate the effect of IL-25 on inflammatory bowel disease,using DSS we established acute colitis model with subsequent treatment of mice with rIL-25 at different concentration so that to be able to determine differential dose effect of IL-25 in the development and progress of acute colitis.Our study revealed inhibitory effect of IL-25 as evidence in the DSS+rIL-25 treated group whereby the mice exhibited delayed development of clinical symptoms including weight loss,diarrhea and rectal bleeding,together with exhibition of less severe colon inflammation of a mild form with low epithelium damage and infiltrate lymphocytes.However,these findings were shown to vary with dosage,suggesting dose dependence inhibition.Cytokine profile in the mice treated with rIL-25 demonstrated that IL-25 reduces the severity of acute colon inflammation by either direct inhibition or suppression of IFN-γwith subsequent induction of IL-10,TGF-Beta and IL-23.Even the lowest dose of IL-25 was sufficient to down-regulate the level of Th type 1 cytokine IFN-γ.IL-25. was found to have no association with IL-17 at the acute stage but the role of IL-4 still remains unknown.Therefore,delay in development of clinical symptoms with less severe colitis observed in the IL-25 treated mice were most likely to be result of a complex cytokine cascade that initiated or associated with IL-25.Despite presence of IL-I7 contents in the mice treated with IL-25,in the present acute colitis investigation we found that neither CD4+/CDS+T cells norγδT cells was shown to be responsible for the production of IL-17 at acute stage if colon inflammation.Conclusions1.DSS-induced ulcerative colitis model is reliable,simple and reproducible model with cytokine profile resemble to human IBD2.IL-25 can successful slow down the development of colitis-induced clinical symptoms and reduce colonic tissue injury and severity of acute colitis in dose dependent manner.3.IL-25 can improve mice survival with full restoration of body weight within short space of time.4.IL-25 expressed is decrease with increase in colitis.5.IL-25 induces IL-10,TGF-β1 and IL-23 in the regulation of acute colitis6.IL-25 show to play crucial role in the regulation of colitis by inhibiting or suppressing Th type 1 cytokine IFN-γ.7.IL-25 possesses anti-inflammatory effect contrary to its fellow family members.